The proposed study will help elucidate the biological role in DNA of the minor base 5-methylcytosine (m5C). Although the average mammalian cell has approximately 4% of its cytosine residues methylated and significantly increased amounts of m5C are found in the DNA of certain cancerous cells, extremely little is known about the influence of methylation of DNA-cytosine on the physiology of normal or cancerous cells. We will study the only known source of DNA in which all of the cytosine is replaced by m5C, bacteriophage XP-12. We have already found that XP-12 DNA has by far the most stable double-stranded structure of any known naturally occurring DNA due to methylation of its DNA cytosine. We will look for XP-12 induced deoxynucleases or RNA polymerases, which can distinguish between m5C-rich DNA (or m5C-rich regions of DNA) and normal, cytosine-rich DNA. These enzymes would be used to specifically hydrolyze the DNA at or preferentially replicate m5C-rich regions in normal DNA. We have already observed higher levels of deoxyribonuclease activity in extracts from cells infected with XP-12 than in those from uninfected cells, and have begun isolation and characterization of these enzymes. If the specific enzymes described above are isolated they will be used as well as standard techniques for base composition analysis and pyrimidine isostich analysis to determine the amount and distribution of m5C in several types of eukaryotic cells. We will compare the results obtained from cultured transformed cells to those from normal cultured cells. Also, we will analyze the DNA from two types of eukaryotic cells at different stages of differentiation, namely, Polysphondylium pallidum undergoing encystment or germination and chick limb bud mesoderm cells undergoing chondrogenesis.